Domestic animal stem cell biology for understanding tissue development, maintenance and therapeutic interventions

马是一种多用途的牲畜，每年为加拿大经济贡献超过190亿美元。肌腱和关节损伤是马类运动员训练天数减少或过早退役的最常见原因之一。干细胞有望为这些难以治疗的损伤提供新的治疗方法。我是一名早期职业研究员（ECR），我的研究计划的总体目标是使用家养动物干细胞作为有效的实验系统来调查和潜在地治疗常见的、经济上重要的牲畜和伴侣动物疾病，同时增强我们对组织再生生物学的基本理解。我实验室的学生将获得各种干细胞群调控的基础知识，在疾病过程的实验研究中培养独特的技能，并在家畜特定骨科疾病中使用这些细胞作为创新的生物工程疗法。本研究项目将重点研究microRNAs （miRNAs）在干细胞稳态、干细胞软骨分化和干细胞免疫调节功能中的作用。mirna是一类内源性非编码rna，被认为参与调节三分之一的哺乳动物基因。今年，我们报道了miR-140在马脐带血源性间充质间质细胞（eCB-MSC）的软骨形成过程中上调。mirna在体内的稳定性和合成模拟物和抑制剂的可用性使它们成为诊断、预后和治疗方法的理想候选者。在接下来的五年里，我将研究特定的mirna在体外调节eCB-MSC软骨形成能力、增强形成的新软骨的生物力学特性和调节宿主动物免疫反应方面的能力。miRNA和mRNA转录组特征将使用下一代测序（NGS）来确定，以发现调节这些过程的新型miRNA，并将miRNA表达与可能的mRNA靶标相关联。基于msc的疗法的发展，战略性地使用选定的miRNA模拟物或抑制剂进行软骨修复和骨关节炎治疗的即时体内测试，以及新的诊断和预后关节生物标志物，预计将从预计的知识收获中获得。这是第一个报道分离出eb - msc的报道。细胞间充质干细胞形成新软骨的能力优于骨髓间充质干细胞和脂肪间充质干细胞。其机制尚不清楚。化学诱导间充质干细胞生成的新软骨力学性能低于正常透明软骨。机械间充质干细胞刺激越来越多地用于复制透明软骨的承重环境。炎症是骨关节炎的主要组成部分。最近在我的实验室中证实了eCB-MSC抑制淋巴细胞增殖的能力，作为抗炎细胞特性的代理。在第一个5年的资助期内，我的主要目标是通过基本的细胞机制研究来补充之前对eCB-MSCs的描述性研究。基于我的项目目标，提出的研究的具体假设是：miRNAs决定了eCB-MSC的软骨细胞和淋巴细胞抑制效力。本提案的3个关键要素是：1)鉴定与MSC高软骨形成效力相关的miRNA特征，并检查所选miRNA在重要靶mrna调控中的作用。2)表征特异性机械反应性mirna在软骨分化中的作用。3)鉴定和表征与eCB-MSCs免疫调节过程相关的mirna。

IntroductionHorses are multi-use livestock that contribute more than $19 billion annually to the Canadian economy. Injuries to tendon and joints are among the most common causes of lost training days or premature retirement in equine athletes. Stem cells hold the promise of novel therapeutic approaches to these difficult-to-treat injuries. I am an Early Career Researcher (ECR) and the overall goal of my research program is to use domestic animal stem cells as potent experimental systems to investigate and potentially treat common, economically important livestock and companion animal diseases while simultaneously enhancing our basic understanding of tissue regeneration biology. Students in my laboratory will gain fundamental knowledge in the regulation of various stem cell populations, develop unique skills in the experimental investigation of disease processes, and in the use of these cells as innovative bioengineered therapies for specific orthopedic conditions in domestic animals. This research program will focus on understanding the role of microRNAs (miRNAs) in stem cell homeostasis, stem cell cartilage differentiation and stem cell immunomodulatory functions. miRNAs are a class of endogenous non-coding RNAs thought to be involved in regulating one third of all mammalian genes. This year we reported that miR-140 is upregulated during chondrogenesis of equine cord blood-derived mesenchymal stromal cells (eCB-MSC). miRNAs in vivo stability and the availability of synthetic mimics and inhibitors makes them ideal candidates for diagnostic, prognostic and therapeutic approaches. I will, in the next five years, interrogate specific miRNAs with regard to their ability to modulate eCB-MSC chondrogenic capacity, enhance biomechanical properties of formed neocartilage, and modulate host animal immune responses in vitro. miRNA and mRNA transcriptome signatures will be determined using next generation sequencing (NGS) to discover novel miRNAs that regulate these processes and correlate miRNA expression to possible mRNA targets. Development of MSC-based therapies, strategic use of selected miRNA mimics or inhibitors for immediate in vivo testing for cartilage repair and osteoarthritic treatments as well as novel diagnostic and prognostic joint biomarkers is expected from the projected knowledge gain. BackgroundI was the first to report the isolation of eCB-MSC. eCB-MSC is better at forming neocartilage than MSC from bone marrow and adipose tissue. The mechanisms are unknown. Neocartilage generated by chemical induction of MSC has inferior mechanical properties compared to normal hyaline cartilage. Mechanical MSC stimulation is increasingly used to replicate the load-bearing environment of hyaline cartilage. Inflammation is a central component of osteoarthritis. The ability of eCB-MSC to suppress lymphocyte proliferation, as a proxy for anti-inflammatory cell properties, was recently confirmed in my lab. ObjectivesDuring this first 5-year funding period, my primary aim is to complement previous descriptive studies on eCB-MSCs with fundamental cell mechanistic investigation. Based on my program goals, the specific hypothesis for the proposed studies is: miRNAs determine eCB-MSC chondrogenic and lymphocyte suppressive potency. The 3 key elements of this proposal are: 1) To identify miRNA signatures associated with high chondrogenic potency of MSC and examine the roles of selected miRNAs in the regulation of important target mRNAs. 2) To characterize the roles of specific mechano-responsive miRNAs in chondrogenic differentiation. 3) To identify and characterize miRNAs associated with immune-modulatory processes in eCB-MSCs.